Wildlife roadkill is a significant threat to biodiversity in Brazil, causing numerous animal fatalities annually. This study aimed to identify the primary locations of wildlife roadkill on two highways passing through Conservation Units (UCs) in Sergipe, examining the correlation between roadkill incidents and periods of drought and rainfall. From November 2021 to October 2022, 49 surveys were conducted along BR-235, near the Serra de Itabaiana National Park, and the Adil Dantas do Amor Cardoso highway, traversing the Mata do Crasto Private Natural Heritage Reserve. Surveys were carried out by motorcycle, traveling at a maximum speed of 60 km/h. To identify roadkill hotspots for each highway, we applied annual mortality indices (MI), Shannon diversity index (H'), species accumulation curves with area extrapolation (Bootstrap), and Kernel density estimation. On BR-235, 63 roadkill incidents were recorded, while on the Adil Dantas do Amor Cardoso highway, which has implemented mitigation measures, there were 15 incidents. No significant variation in roadkill frequency was observed between dry and rainy periods. Areas with mitigation measures experienced lower mortality rates. Determining MI and roadkill hotspots can inform the development of strategies to mitigate the impact of highways on wildlife within conservation areas.